The overall goal of the research is to understand the pathogenesis of intestinal aganglionosis (Hirschsprung disease), which affects 1 in 5000 liveborn humans. The disorder is characterized by congenital absence of nerve cell bodies in the terminal portion of the intestinal tract with consequent obstructive symptoms that usually require surgery. Several murine mutant strains have been developed as models for this condition. In mice and humans, intestinal aganglionosis has been associated with mutations in the RET an ET-B genes which encode receptors expressed by enteric neural precursors. The RET and ET-B receptors are components of intercellular signal transduction pathways that operate during embryogenesis. Mice which lack endogenous endothelin 3 (et3), a ligand for ET-B, are also aganglionic, but can be rescued by transgenic techniques which target expression of et3 to enteric neural precursors. The aims of the proposed research are to determine (1) when and where et-3 expression is required for successful enteric colonization, (2) whether targeted expression of et1 (a homolog of et3) by enteric neural precursors will prevent aganglionosis in et3-deficient embryos, (3) whether abnormal et3 expression plays a role in the pathogenesis of other genetic disorders associated with intestinal aganglionosis (Dom/+, ret-/-) and (4) the effects of activated ras or activated ret expression on intestinal aganglionosis in Dom/+, ret-/- and ET-B-/- mutants. To achieve each of these aims, transgenic techniques will be used to regulate spatiotemporal expression of the aforementioned gene products in wild-type and mutant embryos with the hope of altering the mutant phenotype. Presence or absence of aganglionosis will be analyzed histologically and immunohistochemically. The data generated will define spatial and temporal constraints on et3 production in embryos that exist for complete colonization of the gut by enteric neural precursors. In addition, in vivo evidence for interactions between RET, ET-B an on of their intracellular effectors, ras, wil be sought in an effort to clarify how mutations which affect these two signal transduction pathways lead to a similar birth defect. The information gained from these studies wil serve as a foundation for the development of more efficient methods to diagnose, prevent, and perhaps treat Hirschsprung disease.